Sleep loss produces deficits in hippocampal synaptic plasticity and hippocampus-dependent memory storage. However, the molecular and cellular mechanisms that underlie these effects of sleep deprivation remain unclear. Our recent work suggests that sleep deprivation alters protein synthesis, reducing translation initiation. In this application, two Specific Aims are proposed that use novel genetic approaches to modulate hippocampal eukaryotic initiation factor-binding protein 2 (4EBP2) expression to study how sleep deprivation affects translation and to identify the molecular consequences that impact cognition. One pathway that regulates protein translation is mammalian target of rapamycin complex 1 (mTORC1) signaling, which acts through two downstream pathways, S6 kinase and 4EBP2. Our published work and preliminary studies show that sleep deprivation attenuates mTORC1 signaling leading to reduced total and phosphorylated levels of 4EBP2. However, it is not known whether these sleep deprivation-induced changes in 4EBP2 expression have functional effects in the hippocampus. In Specific Aim 1, genetic techniques will be used to determine the role of 4EBP2 and hippocampal protein translation initiation in the memory and plasticity deficits observed after sleep deprivation. The mechanism by which translation initiation is impaired with sleep deprivation will be investigated in Specific Aim 1, but the molecular consequences of impaired translation has yet to be characterized. Therefore, in Specific Aim 2A we will use novel genetic tools that utilize recently developed RNA tagging and recovery of associated proteins (TRAP) technology to selectively isolate ribosome-bound mRNA from excitatory pyramidal neurons of the hippocampus. We will employ next generation sequencing to identify the messenger RNAs that are undergoing active translation during sleep deprivation to define the molecular changes caused by sleep deprivation. In Specific Aim 2B, these newly identified targeted mRNAs will be validated in the 4EBP2-AAV mice from Specific Aim 1 to determine which specific protein expression is restored with rescued memory and translation. Sleep deprivation is a significant public health issue in our society with millions of people obtaining insufficient sleep that greatly affects quality of life. Additionally, many people who hae psychiatric and neurodegenerative disorders, including depression, schizophrenia, and Alzheimer's disease, also suffer from sleep loss. This proposal seeks to reverse the negative impact of sleep deprivation on translation and its subsequent impairments in cognition. Knowledge of the cellular mechanism and molecular consequences associated with sleep loss promises to lead to the development of novel therapeutic approaches to alleviate the cognitive deficits that result from sleep deprivation.